003 1-3998/90/2804-003 16$02.00/0 PEDIATRIC RESEARCH Vol. 28, No. 4, 1990 Copyright 0 1990 International Pediatric Research Foundation, Inc Printed in U.S.A. Development of Natural Killer Cytotoxicity during Childhood: Marked Increases in Number of Natural Killer Cells with Adequate Cytotoxic Abilities during Infancy to Early Childhood AKIHIKO YABUHARA, HIROSHI KAWAI, AND ATSUSHI KOMIYAMA Department of Pediatrics, Shinshu University School of Medicine, Mastumoto, Japan ABSTRACT. The cytotoxicity of natural killer (NK) cells virus exposure of neonates can result in severe infection, which against K562 cells and their responsiveness to interferon- is explained by the depressed activity of NK cells (5-8). Although a and interleukin 2 (IL-2) were studied throughout child- viral infections cause various clinical pictures at different levels hood using S'Cr-release and single-cell assays. Although of ontology (9), very little is known about the developmental NK activity was extremely low in the neonatal period, it profile of NK cytotoxicity during childhood. almost reached the adult level during 1 to 5 mo of age and It is known that the number of lymphocytes in the peripheral remained at that level thereafter. At the single-cell level, blood varies widely among different ages of childhood. Never- the binding, lytic, and recycling abilities were also de- theless, NK activity usually represents the cytotoxicity of a given pressed in the neonatal period, but these abilities improved number of lymphocytes (2). Although morphologic examination conspicuously after this period; in particular, the lysis and and surface marker analysis have been commonly used to deter- recycling were at higher levels during 6 mo to 4 y of age. mine the number of NK cells (2, lo), there have been conflicting The absolute numbers of circulating cytotoxic NK cells reports regarding the relationship between the NK cell number were high during infancy to early childhood: they were 54 and NK activity; the percentages of large granular lymphocytes f 24 (mean f SD/mm3) in neonates, 115 f 48 in 1- to 5- and CD16' cells in neonates are similar to those in adults (1 1, mo-old infants, 121 + 42 in 6- to 12-mo-old infants, 93 + 12),but NK activity is depressed in neonates (6, 13, 14). A single- 26 in 1- to 4-y-old children, and 42 f 16 in adults. Inter- cell cytotoxicity assay allows the determination of the number feron-a and IL-2 could enhance NK activity throughout of NK cells with cytotoxic abilities (2, 15). childhood. The IL-2 enhancement was prominent espe- The purpose of our study was to investigate the developmental cially in the neonatal period; IL-2 yielded a 2.5-fold in- profile of the cytotoxicity mediated by circulating NK cells crease in the number of cytotoxic cells and improved the during childhood using 51Cr-releaseand single-cell assays. In our recycling to the adult level. At older ages, interferon-a and study, we demonstrate the normalization of NK cell functions IL-2 yielded 1.4- and 1.9-fold increases in the number of after the neonatal period and an increased number of the cells cytotoxic cells, respectively, but did not enhance the recy- during infancy to early childhood. cling. The increased number of NK cells with adequate cytotoxic abilities during infancy to early childhood indi- MATERIALS AND METHODS cates the predominance of NK immunity during these periods. IL-2 is a cytokine that induces high levels of NK Subjects. Cord blood samples were collected immediately after cytotoxicity even in neonates. (Pediair Res 28: 316-322, delivery of normal full-term newborns; all babies were born of 1990) healthy mothers with uncomplicated pregnancy and delivery. The blood of full-term newborn infants was obtained from the peripheral artery when partial exchange transfusions were per- Abbreviations formed during 1 to 3 d of life because of polycythemia; these infants were also born of healthy mothers with uncomplicated NK, natural killer pregnancy and did not have an associated condition such as IFN, interferon Down's syndrome or congenital adrenal hyperplasia. Peripheral IL-2, interleukin 2 venous blood samples were obtained from children ranging from E:T, effector:target 1 mo to 13 y of age who visited our hospital for examination for % TBC, percentage of target-binding cells some immunodeficiency but proved to be free, and from healthy % DC, percentage of dead conjugates adults ranging from 2 1 to 30 y of age. MRC, maximal recycling capacity Preparation of lymphocytes. Mononuclear cells were separated from the heparinized blood by Ficoll-Hypaque gradient centrif- ugation and depleted of adherent cells by incubation on plastic culture dishes. The nonadherent cells were resuspended in RPMI- 1640 medium (Flow Laboratories, Irvine, Scotland) con- Growing evidence indicates that NK cytotoxicity is a response taining 10% heat-inactivated FCS and used as lymphocytes in to viral infection before the generation of antiviral antibody and this study. cytotoxic T lymphocytes (1-3) and NK cells play an important Treatments oflymphocytes with IFN-a andIL-2. Lymphocytes role in the early protection against primary infections (4, 5). The (1 x 106/mL) were incubated for 24 h in a humid atmosphere of 5% C02 at 37°C with medium alone, 1000 U/mL IFN-a Received December 19, 1989; accepted May IS, 1990. Correspondence: Dr. Akihiko Yabuhara, Department of Pediatrics, Shinshu (K~OwaHakkO CO., Or 200 U/mL University School of Medicine, Asahi 3-1-1, Matsumoto, 390, Japan. IL-2 (Takeda Seiyaku Co., Osaka, Japan). After incubation, the 316 DEVELOPMENT OF NK CYTOTOXICITY 317 cells were washed once and used as effector cells in cytotoxicity values (r = 0.959). Therefore, it was possible to get rough assays. estimation of the V,,, values using the "Cr-release assay. The Assay for NK activity. NK activity was measured in a 4-h 5'Cr- V,,, values were calculated using the following formula: V,,, = release assay using K562 target cells as previously described (16). 1.9 X lo3 + 4.0 X 10' x % cytotoxicity in 5:l. Briefly, effector cells were mixed with 51Cr-labeledK562 cells (1 Single-cell cytotoxicity assay in agarose. This assay was per- x lo4) in a total volume of 200 pL in the wells of round- formed according to the method of Ullberg and Jondal(15) with bottomed microtiter plates (Nunc, Roskilde, Denmark) at an some modifications (17). Briefly, equal numbers of effector cells E:T ratio of 5: 1 or 20: 1, and the cells were incubated for 4 h at and unlabeled K562 target cells (2 x lo5) were mixed in a total 37°C in a 5% COz atmosphere. After incubation, the plates were volume of 200 pL RPMI-1640 with 10% FCS, centrifuged at 250 centrifuged, and 100 pL of the supernatant were harvested and x g for 5 min, and incubated at 37°C for 10 min. The resulting assayed for radioactivity. NK activity, expressed as % cytotoxic- pellet was gently resuspended by Pasteur pipetting 10 times and ity, was calculated by the following formula: % cytotoxicity = the resuspended cells were then carefully added to 0.5% agarose [(experimental release - spontaneous release)/(total release - in RPMI-1640, which had been precooled from 48°C to 39°C. spontaneous release)] x 100. The cells were mixed with a Pasteur pipette and immediately Estimation of I/,,,. The maximum NK potential (V,,,) of poured onto culture plates precoated with 0.5% agarose. After effector cells was determined by a modification of the method of solidifying, the plates were overlayered with culture medium and Ullberg and Jondal (15). Inasmuch as the dose-response curve incubated at 37°C for 4 h. After the medium was removed, the from the "Cr-release assay resembled Michaelis-Menten enzyme- plates were stained with 0.1 % trypan blue for 5 min, washed substrate kinetics, the V,,, values could be determined using with PBS, and fixed with 1% formaldehyde. Control plates of Lineweaver-Burk plots. Briefly, lo5 effector cells in 100 pL were target cells alone were prepared in an identical manner. incubated for 4 h in round-bottom microtiter wells with 100 pL The plates were examined under a microscope. The % TBC of "Cr-labeled K562 cells at six doubling dilutions, from 4 x lo5 was determined by counting the number of lymphocytes bound to 1.25 x lo4 cells per well. The values of % cytotoxicity were to target cells in 400 lymphocytes. The percentage of dead target calculated for each target cell concentration as described above. cells in the conjugates was determined by counting the number The number of killed target cells was determined by multiplying of trypan blue-positive target cells in 100 conjugates. The per- the initial number of target cells by % cytotoxicity. The V,,, of centage of spontaneously dead target cells was determined by lo5 effector cells was determined from a linear regression curve counting the number of trypan blue-positive cells in 100 target obtained by plotting the reciprocals 1/V (ordinate) and 1/T cells on control plates. Corrections were made using the following (abscissa), where V is the number of killed target cells and T is formula to calculate the % DC: % DC = (percentage of dead the initial number of target cells. The V,,, was obtained from target cells in conjugates) - (percentage of spontaneously dead the reciprocal of the intercept of the curve with the ordinate and target cells) x (percentage of dead target cells in conjugates). represents the maximum number of target cells that can be killed Estimation of percentage of cytotoxic cells and MRC. The by lo5 effector cells (Fig. 1A). percentage of cytotoxic cells was estimated from the single-cell Subsequently, we simplified this procedure by finding that the cytotoxicity assay by multiplying % TBC by % DC.